Photosynthetic responses of Larix kaempferi and Pinus densiflora seedlings are affected by summer extreme heat rather than by extreme precipitation

The frequency and intensity of summer extreme climate events are increasing over time, and have a substantial negative effect on plants, which may be evident in their impact on photosynthesis. Here, we examined the photosynthetic responses of Larix kaempferi and Pinus densiflora seedlings to extreme heat (+ 3 °C and + 6 °C), drought, and heavy rainfall by conducting an open-field multifactor experiment. Leaf gas exchange in L. kaempferi showed a decreasing trend under increasing temperature, showing a reduction in the stomatal conductance, transpiration rate, and net photosynthetic rate by 135.2%, 102.3%, and 24.8%, respectively, in the + 6 °C treatment compared to those in the control. In contrast, P. densiflora exhibited a peak function in the stomatal conductance and transpiration rate under + 3 °C treatment. Furthermore, both species exhibited increased total chlorophyll contents under extreme heat conditions. However, extreme precipitation had no marked effect on photosynthetic activities, given the overall favorable water availability for plants. These results indicate that while extreme heat generally reduces photosynthesis by triggering stomatal closure under high vapor pressure deficit, plants employ diverse stomatal strategies in response to increasing temperature, which vary among species. Our findings contribute to the understanding of mechanisms underlying the photosynthetic responses of conifer seedlings to summer extreme climate events.

(H1) The extreme heat treatment would decrease g s in L. kaempferi and P. densiflora by inducing stomatal closure under high VPD L (H1a), and this decrease would be most pronounced under concurrent drought treatment (H1b).Additionally, the treatments of extreme climate events would decrease chlorophyll contents (H1c).(H2) As a consequence of decreased g s , E and P n would decrease under the extreme heat, drought, and heavy rainfall treatments.

Experimental design
The open-field experiment was carried out in Pocheon, South Korea (37° 45′ 38.9″ N, 127° 10′ 13.4″ E) (Fig. S1).This site is in the humid continental climate zone, characterized by hot summers and cold/dry winters 29 , with a high inter-annual variation of annual precipitation.Over a period of 23 years (1997-2019), the mean annual temperature at this site ranged from 9.2 to 11.4 °C and the annual precipitation ranged from 870 to 2329 mm.The experimental setup consisted of six blocks, within each of which nine 1.5 m × 1.0 m plots were established (Fig. S2).Three blocks were assigned for L. kaempferi and the remaining three blocks were designated for P. densiflora.Within each plot, a random combination of two types of treatments was assigned: temperature treatments (ambient, ambient + 3 °C, and ambient + 6 °C; referred to as TC, T3, and T6, respectively) and precipitation treatments (ambient, complete exclusion of rainfall as extreme drought, and water addition above the ambient as heavy rainfall; referred to as PC, DR, and HR, respectively).Two factorial combinations were introduced, consisting of three temperature regimes and three precipitation regimes.Consequently, 54 plots were arrayed at the experimental site (two species × three temperature levels × three precipitation levels × three replicates).In April 2020, a total of 88 and 99 1-year-old L. kaempferi and P. densiflora seedlings, respectively, were planted in each plot following the guidelines for seedling management provided by Korea Forest Service (2020).The soil texture at the experimental site was classified as sandy loam (70% sand, 20% silt, and 10% clay).
Infrared heaters (FT-1000, Mor Electronic Heating Assoc., Comstock Park, MI, USA) were used to increase the canopy temperature (CT) in the T3 and T6 treatments (Fig. S4).Infrared thermometers (SI-111, Apogee Instruments, Logan, UT, USA) measured the CT of experimental plots, and dataloggers (CR1000X, Campbell Scientific, Inc., Logan, UT, USA) and relays (SDM-CD-16AC, Campbell Scientific, Logan, UT, USA) maintained the target temperature under the T3 and T6 treatments (if CT reached the target temperature, relays switched off the heaters).To monitor soil temperature (ST) and SWC, measurements were taken at a depth of 5 cm using a soil temperature/moisture sensor (SI-111, Campbell Scientific, Logan, UT, USA).An automatic rainout shelter with a transparent roof (2.0 m × 1.5 m) intercepted the natural rainfall in DR plots.The rainout shelter would close only when a rain detector (HTL-301, Haimil, Republic of Korea) detected rainfall, in order to avoid disturbance from the microclimate (e.g., light and airflow) within the plots.For the HR treatment, an artificial rainfall simulator was used.This simulator employed two spray nozzles (Unijet D5-35, Spraying Systems Co., Wheaton, IL, USA) per plot, spraying water stored in a tank.For more detailed information on the experimental design, please refer to Fig. S3 and the study conducted by Kim et al. 30 .
To determine the threshold and establish the experimental scenario of extreme climate events, we utilized meteorological data from the reference period of 1961-2019 for the months of July and August in Seoul.Since meteorological data for the research site were available only after 1997, we used the data from the nearest city, Seoul, located approximately 30 km away, as a reference (Fig. S1).The target temperatures of T3 and T6 were determined based on the difference between the mean daily maximum temperature (29.9 °C) and the 90th (33.2 °C) and 99th (36.0 °C) percentiles of the daily maximum temperature, respectively, during the reference period 1 .The duration of the extreme heat treatment was determined as the longest period of consecutive days with a daily maximum temperature above the threshold for extreme heat during the reference period, which was determined to be 7 days.For DR, it was defined as the longest period of consecutive days with daily precipitation of less than 1 mm during the reference period, which amounted to 9 days 31 .HR was defined as the 95th percentile of the daily precipitation during the reference period, which amounted to 113 mm day −132 .To determine the duration of HR, we calculated the longest consecutive period with daily precipitation exceeding the threshold of heavy rainfall, which was set at 3 days.
We simulated these extreme climate events from July-August 2020.The manipulation of temperature and precipitation was divided into two periods, with one-week of no treatment period in-between the treatment periods.The first and second DR treatments were applied on the 195-203 and 218-226 day of the year (DOY), respectively.However, during the second period, soil water content (SWC) in DR plots unexpectedly increased due to naturally occurring heavy rainfall

Measurement of photosynthetic parameters
During the experimental period of July-August 2020, in situ measurements of leaf gas exchange of L. kaempferi and P. densiflora seedlings were performed using a portable photosynthesis system (LI-6800, Li-Cor Inc., Lincoln, NE, USA) with a 3 cm × 3 cm chamber (6800-12A, Li-Cor Inc., Lincoln, NE, USA).Leaf gas exchange measurements were conducted five times throughout the experimental period.The measurements were carried out on the needles of three randomly selected seedlings per each plot.The measurements were taken at a photosynthetic photon flux density of 1000 µmol m −2 s −1 , relative humidity of 50%, CO 2 concentration of 400 µmol mol −1 , and an ambient air temperature ranging from 27 to 33 °C.Consistent with the experimental design, gas exchange measurements were conducted between 0900 and 1500 h to minimize any diurnal variations.After field measurements were conducted, the needles were brought to the lab, where their area was determined using a scanner (Perfection V700 Photo, EPSON, Japan) and an image analysis system (WinSEEDLE, Regent Instruments Inc., Québec City, QC, Canada).This information was used to calculate the measured values of P n , E, g s , and the ratio of intercellular to ambient CO 2 concentration (C i /C a ) on a leaf area basis.Additionally, water use efficiency (WUE) and intrinsic water use efficiency (iWUE) were calculated using the ratios of P n /E and P n /g s , respectively.
To measure the chlorophyll content, the needles were cut to a length of approximately 2 mm.Subsequently, 20 ± 1 mg of cut needles were placed into vials containing 5 mL of dimethyl sulfoxide (DMSO).The vials were then incubated at 65 °C for 6 h in a water bath (HQ-DW22, Coretech Korea Co., Republic of Korea) to extract the chlorophyll.After the incubation period, the absorbance of the chlorophyll extracts was measured at 648 nm and 665 nm using a spectrophotometer (UH5300, Hitachi, Japan).The absorbance values at these wavelengths were used to calculate the chlorophyll a (Chl a ), chlorophyll b (Chl b ), and total chlorophyll (Chl t ) contents using the following equations 33 : where A 665 and A 648 are the absorbances at 665 nm and 648 nm, respectively.V is the volume of DMSO, and F.W. is the fresh weight of needles.
To calculate VPD L , we derived hourly air temperature and relative humidity from the automatic weather station at the experimental site.The leaf temperature for VPD L calculation was obtained from the infrared thermometer in the plots.VPD L was calculated based on air saturation vapor pressure (ASVP) and leaf saturation vapor pressure (LSVP) using the following equations 14,34 : (1) Chl a = (14.84× A 665 −5.14 × A 648 ) × V ÷ F.W.  where T air and T leaf are the air and leaf temperatures, respectively, and RH is relative humidity.We assumed that all plots were under the equivalent RH, considering that the experiment was conducted in an open field 35 .

Data analysis
The effects of temperature and precipitation manipulation on the environmental factors were examined using repeated measures analysis of variance (ANOVA).Additionally, the effects of temperature and precipitation manipulation on the VPD L , leaf gas exchange, and chlorophyll content were determined by two-way ANOVA using a linear mixed model to account for a randomized complete block design.The block was treated as a random effect and the temperature and precipitation treatments were treated as fixed effects.The linear mixed model equation used for analysis is as follows: where Y ijkl is the response variable in the ith observation (i = 1-5) under jth temperature treatment T (j = TC, T3, or T6) and kth precipitation treatment P (k = PC, DR, or HR) with lth block (l = 1-3).TP is the interaction between T and P, β 0 is the intercept, β n are coefficients to be estimated (n = 1-3), ε k is the random residuals associated with the block, and ε ijkl is the final residuals.As coefficients of P and TP were not statistically significant for all photosynthetic parameters (P > 0.05), the variables were removed from the analysis.In addition, due to an increase in SWC in the DR treatment during the second period, caused by naturally occurring heavy rainfall, we excluded the data on photosynthetic activities measured during and following this period from the analysis.We verified the statistically significant differences among precipitation treatments within temperature treatments via Tukey's post hoc test.
The effect size for each parameter was calculated as the natural logarithm of the response ratio (RR) to compare the means of treatment (T3, T6, DR, or HR) with the means of control (TC or PC) by the following equation 36 : where X t and X c are the means of parameters in the temperature and precipitation treatment and control, respectively.The variance (v) of RR was calculated as: where SD t and SD c are the standard deviation of parameters in the treatment and control, respectively, and n t and n c are the sample sizes of parameters in the treatment and control, respectively.The statistical significance between the treatment and control was determined by Tukey's post hoc test.
The relationships among VPD L , E, C i /C a, and P n with g s were examined by non-linear regression.Specifically, the relationship between VPD L and g s was determined using the equation proposed by Oren et al. 37 : where m represents the slope and b represents a reference g s at VPD L = 1 kPa.
Principal component analysis (PCA) was carried out to determine the relationships among environmental factors and photosynthetic activities.All data analyses and visualizations were conducted with R version 4.2.1 at a significance level of 0.05 38 .R packages of "lme4" for linear mixed model 39 , "sjstats" for RR 40 , and "ggplot2" for data visualization 41 were used.

Research ethics
The experimental research on L. kaempferi and P. densiflora seedlings, including the collection of seedling material, complied with relevant institutional, national, and international guidelines and legislation.As the seedlings were cultivated and maintained by the National Institute of Forest Science, a joint research institute involved in this study, no specific permissions were required for the seedling collection.

Environmental conditions
CT during the temperature manipulation period was significantly different among TC, T3, and T6 (P < 0.001) (Fig. 1a).The mean CT during the first temperature manipulation period was 2.6 °C and 5.8 °C higher in T3 and T6, respectively, compared to that in TC.During the second period, the mean CT in T3 and T6 was also significantly higher (2.6 °C and 5.7 °C) than that in TC, respectively (P < 0.001).Temperature manipulation also significantly affected ST during both temperature manipulations (P < 0.001) (Fig. 1b).The mean ST (°C ± one standard error) was 22.7 ± 1.0, 24.5 ± 1.2, and 26.3 ± 1.8 in TC, T3, and T6, respectively, during the first temperature manipulation period.During the second period, ST was 26.0 ± 0.4, 27.3 ± 0.5, and 28.9 ± 0.9 in TC, T3, and T6, respectively.
There was no significant effect of temperature manipulation on SWC (P = 0.59) (Fig. 1c).Precipitation manipulation significantly affected SWC during the first manipulation period (P < 0.001).The mean SWC (vol.%) during this period was 7.8 ± 2.2, 10.1 ± 2.0, and 12.2 ± 2.1 in DR, PC, and HR, respectively.The difference in SWC among treatments during the second manipulation was also statistically significant (P = 0.04).The mean ( 6)

Leaf gas exchange and chlorophyll content
The VPD L significantly increased with increased temperature in both L. kaempferi and P. densiflora (P < 0.001) (Fig. 2a,b).VPD L ranged between 1.10-1.67kPa in L. kaempferi and 1.11-1.70kPa in P. densiflora.g s significantly decreased as VPD L increased in L. kaempferi (P < 0.001) (Fig. 2c), whereas there was no significant correlation between g s and VPD L in P. densiflora (P = 0.37) (Fig. 2d).
By analyzing all photosynthetic parameters and environmental factors with PCA, the biplot of L. kaempferi showed a definite grouping by temperature treatments (Fig. 7a).PC1 of L. kaempferi explained 56.38% of the variations and indicated that E, g s , and C i /C a were negatively related to CT, WUE, and iWUE.PC2 explained 14.54% of the total variations and indicated the positive correlation among P n , Chl t , CT, and ST.The results of PCA for P. densiflora seedlings showed that PC1 and PC2 explained 54.30% and 17.61% of the total variations, respectively (Fig. 7b).PC1 revealed that E and C i /C a were negatively related to the WUE and iWUE of P. densiflora and PC2 explained the relationship among environmental factors.

Discussion
The functionality of temperature and precipitation manipulation systems is crucial when conducting experiments in the open field, as ambient climate factors can easily influence the experimental treatments 2 .In this study, the temperature manipulation system successfully simulated the conditions of real extreme heat events.The temperature manipulation system ensured that the heated and ambient plots had distinct CT and ST conditions, even during periods of rainfall (DOY 205 and 228) or under extremely high air temperature (33.1 °C on DOY 232; data not shown) conditions.However, there was an unexpected increase in SWC in DR plots during the second manipulation period.These results were inconsistent with those that would occur under water stress, and we propose that the cause of the increased SWC was the naturally occurring extreme precipitation events.Specifically, during the rest period between the two periods of experimental precipitation manipulation, the study area received a significant amount of rainfall (358 mm) over a five-day period (DOY 213-217), which accounted for 26% of the mean annual precipitation over 23 years.It is suggested that the rainfall may have entered the plots through open sides, resulting in an increase in SWC in DR plots.These unexpected results emphasize a limitation commonly associated with the open-field experiment.Thus, we suggest considering the edge part of the plots as a buffer zone to minimize the potential influence of ambient factors, such as lateral influx of rainfall or microclimate variations when conducting the open-field experiment.
Consistent with our hypothesis (H1a), as the temperatures increased, g s in L. kaempferi showed a decreasing trend (Fig. 3a).This result is consistent with the concept that high temperatures can lead to stomatal closure in order to mitigate water loss 14,42 .Elevated evaporative demand and subsequent leaf water loss associated with high temperatures may also contribute to stomatal closure 18,43 .The significant negative relationship between g s and VPD L in L. kaempferi found in this study was supported by these established theories (Fig. 2c).This decreasing trend in g s under high temperatures and VPD L confirms the findings of a previous study by Ameye et al. 44 wherein the photosynthetic responses of P. taeda seedlings were examined under experimental heat waves with a biweekly + 6 °C treatment.The closure of stomata is likely responsible for the reduction in E and C i /C a 14, 15,45,46   .In our study, the positive relations observed between E, C i /C a , and g s in L. kaempferi as well as PCA results provide further evidence that the decrease in E and C i /C a is associated with stomatal closure, thus in line with H2.The decrease in CO 2 uptake can lead to oxidative damage and a decline in P n 47,48 . Additionally, heat stress may inhibit the CO 2 fixation of plants and damage components of their photosynthetic apparatus, especially photosystem II, which plays a crucial role in electron transport during photosynthesis, as leaf temperature increases 47 .However, in our study, P n of L. kaempferi significantly decreased only under T6, but not under T3.This result suggests that the relatively lower VPD L under T3, compared to T6, was not sufficient to inhibit P n , since the sensitivity of P n to VPD L is weaker than that of g s 14 .In contrast to L. kaempferi, the increasing VPD L did not have a significant impact on g s of P. densiflora (Fig. 2d).Furthermore, g s and E of P. densiflora increased under T3 and decreased again in T6 (Fig. 5c).These results are contrary to H1a, suggesting species-specific variation of stomatal behaviors in responses to temperature and VPD L .The observed peak responses of g s and E to increasing VPD L and temperature can be interpreted as a trade-off between the 'feed-back' and 'feed-forward' stomatal responses.Stomatal transpiration can increase as a strategy for cooling the leaf surface under high temperatures, representing a feed-back response 49 .Conversely, a feed-forward response involves a decline in E as temperature and VPD L increase to avoid hydraulic failure 43,50 .During the feed-back response, the evaporative cooling strategy may induce water loss through the leaf cuticle.Subsequently, g s and E may begin to decrease after reaching a certain level of VPD L and temperature in response to this water loss, aiming to prevent hydraulic failure, thus exhibiting a peak function 14,51,52 .Although these peaked responses have been a subject of debate as it is difficult to explain the response from simple stomatal mechanisms, previous studies have suggested that there is an optimum VPD L and temperature for E 51 .Furthermore, previous findings have observed that these responses are more likely to occur when temperature co-varies with VPD L , rather than when temperature remains constant.The observed peaked response of g s and E of P. densiflora under the extreme heat treatment can be interpreted as a result of the trade-off between the evaporative cooling and minimizing water loss, particularly given the covariation of temperature and VPD L in our experiment.
These divergent stomatal strategies in response to extreme heat may be attributed to differences in the species' hydraulic traits 14 .Specifically, the high sensitivity of g s to VPD L in L. kaempferi suggests an isohydric behavior.In contrast, P. densiflora appeared to withstand the extreme heat stress through anisohydric stomatal regulation, as evidenced by its consistent P n under the treatments.In addition, the distinction in needle morphology could   contribute to variation in stomatal behaviors between the two species.Longer needles are likely to receive a higher irradiance on their surface, resulting high requirement for CO 2 uptake, compared to shorter needles 53,54 .This demand is met through increased leaf hydraulic conductance, g s , and evaporative demand.Furthermore, longer needles possess a higher hydraulic capacity, essential for delivering the water needed to maintain open stomata 53 .These hydraulic traits of a long leaf are evidenced by higher values of g s , E, and P n , and feed-back stomatal response in P. densiflora in this study which has a longer needle (approximately 5.93 ± 0.23 cm needle −1 ) compared to L. kaempferi (approximately 2.31 ± 0.05 cm needle −1 ).Additionally, the evergreen characteristics of P. densiflora likely contribute to the differential stomatal behavior, as evergreen species may invest more in carbon uptake due to their longer leaf lifespan, in contrast to deciduous species such as L. kaempferi, which   www.nature.com/scientificreports/have 'disposable' leaves 55 .The distinct hydraulic regulations might be a critical factor in plant mortality under environmental stresses 56 .Anisohydric behavior may provide short-term tolerance to heat stresses, as observed in our study.However, the prolonged hot and dry conditions can rapidly lead anisohydric species to dehydration and xylem cavitation, ultimately resulting in mortality due to their increased E at high temperatures 16 .Therefore, examining photosynthetic activities under long-term environmental stresses is essential for predicting plant survival following extreme climate events.Interestingly, P n in P. densiflora did not show changes with increasing temperature and there was no observed correlation between P n and g s (Figs.3h, 6f).These findings suggest that P n in P. densiflora may be influenced by factors other than g s , indicating a more complex relationship between stomatal behavior and photosynthetic performance.A study by Urban et al. 22 examining gas exchange variables in responses to increasing temperature in P. taeda and Populus deltoides x nigra also observed the decoupled relationship between g s and P n at leaf temperatures > 40 °C, further supporting the complex interactions between these variables.In addition, a peak response of gs to increasing VPD L was likely a contributing factor to the decoupling between g s and P n 57 .These results highlight the need for further research to explore underlying mechanisms influencing P n in P. densiflora and to elucidate the factors that contribute to its photosynthetic response under extreme temperature conditions.
Contrary to our hypothesis (H1c), our study found markedly high chlorophyll contents under the extreme heat treatment for both L. kaempferi and P. densiflora.While chlorophyll contents generally tend to decrease under thermal stress due to leaf senescence, there may exist an optimal temperature range where chlorophyll contents can increase as temperature rises.Previous studies have found that high temperatures may enhance plant growth and accelerate pigment biosynthesis, and the activity of enzymes involved in chlorophyll production, resulting in an increase in chlorophyll contents 58,59 .However, upon examining the growth and biomass data from the current study (data not shown), the temperature treatments did not have an impact on seedling growth and biomass in either species.Seedling height (cm) and total biomass (g seedling −1 ) of L. kaempferi and P. densiflora were not affected by the temperature treatment (P = 0.27 and 0.88, respectively, for L. kaempferi; Noh et al. 26 , and P = 0.61 and 0.85, respectively, for P. densiflora; unpublished data), ranging from 48.6-57.5 and 13.24-15.88,respectively, in L. kaempferi, and from 28.6-29.7 and 6.89-7.96,respectively, in P. densiflora.Consequently, the increased chlorophyll contents in the extreme heat treatment might be attributed to the accelerated pigment biosynthesis rather than the enhanced growth of seedlings.Yun et al. 27 also observed that Chl t in P. densiflora increased within the air temperature range of 15-31 °C in their experiment, where an increase in air temperature by 3 °C was simulated using the infrared heater.In our study, the observed high chlorophyll contents under extreme heat, with CT ranging from 24 to 32 °C, suggest that this temperature range may correspond to the optimum conditions for pigment biosynthesis.
In general, water deficit conditions typically lead to stomatal closure and a reduction in CO 2 uptake, thereby reducing g s , E, and P n 60,61 .However, in this study, we did not observe a decrease in photosynthetic parameters under DR in both L. kaempferi and P. densiflora (Fig. 5b,d).This result could be attributed to the brief duration of summer drought spells experienced in the East Asian monsoon climate of the study region.While the lack of rainfall per se can be considered "extreme", the brief nature of these drought spells may not have been sufficient to evoke significant changes in the photosynthetic activities.In addition, the naturally occurring extreme rainfall during the rest period likely counteracted the effect of water treatments by considerably raising the water availability level throughout the site.These findings highlight the importance of considering the dynamic nature of rainfall patterns in the East Asian monsoon regions, as summer rainfall events can provide adequate SWC for sustaining photosynthetic activities, mitigating the negative effects of summer extreme drought conditions.Consequently, although the SWC in HR was significantly higher than that in DR and PC, the difference did not translate into photosynthetic responses to water treatments.The natural rainfall during the rest period and the second manipulation period seemed to have already produced enough SWC in PC, which met the water demand for photosynthesis, regardless of HR treatment.In a study investigating the mechanisms linking increased rainfall and water dynamics, Lopez et al. 62 found that the crown E of L. cajanderi in an irrigated plot (under SWC of 25-28 vol.%) did not differ substantially from that in the ambient plot (under SWC of 15-25 vol.%).Similarly, Jo et al. 63 did not observe notable differences in the P n , E, and g s of Abies holophylla and A. koreana when the SWC increased from approximately 15 to 25 vol.%.Moreover, due to the soil texture (sandy loam) in this study, which has a relatively low water holding capacity 64 , it is unlikely that the difference in SWC between HR and PC plots persisted for a long period of time after the HR treatment.

Conclusion
To summarize, we found that L. kaempferi showed a decrease in g s under extreme heat, leading to the reduction in all photosynthetic parameters, whereas P. densiflora showed a peak function in g s and E under extreme heat and no change in P n .No effect was observed of extreme drought and heavy rainfall on photosynthetic activities in both species.These findings reveal the species differences in stomatal behaviors in response to increasing temperature and L. kaempferi experiencing more pronounced adverse effects on photosynthesis compared to P. densiflora.These results indicate that extreme heat may have a more negative impact on forest succession dynamics and degrade ecosystems' function in newly established L. kaempferi forests, by inhibiting carbon uptake, in comparison to P. densiflora forests.Therefore, these findings suggest the significance of implementing temperature management strategies in nursery systems, particularly for L. kaempferi, to effectively respond to extreme climate events.However, we note that the observed responses spanned only a single season, whereas experimental treatments in open-field trials can be inferred by natural environmental stochasticity, necessitating a long-term study.Thus, further long-term studies are needed to assess the lagged effect of summer extreme conditions in subsequent seasons and the recovery capacity of plants from extreme climate events.

Figure 1 .
Figure 1.Canopy temperature (CT) (a), soil temperature (ST) (b), and soil water content (SWC) (c) and daily precipitation (gray bars) during the experimental period.Red and blue areas mean the period of temperature and precipitation manipulation, respectively.TC: temperature control; T3: + 3 °C treatment; T6: + 6 °C treatment; DR: extreme drought; PC: precipitation control; HR: heavy rainfall treatment.Asterisks depict statistical differences among TC, T3, and T6 in CT and ST, and DR, PC, and HR in SWC (*P < 0.05; **P < 0.01; ***P < 0.001).DOY means day of the year.This figure was modified from Kim et al. 30 .

Figure 5 .
Figure 5.The effect size of photosynthetic parameters of Larix kaempferi (a, b) and Pinus densiflora (c, d) seedlings.Yellow and red colored-symbols indicate the + 3 °C treatment (T3) and + 6 °C treatment (T6), respectively, and orange and blue-colored symbols indicate the extreme drought (DR) and heavy rainfall (HR) treatment.Horizontal lines represent one standard error.Asterisks show the significant differences to the control (*P < 0.05; **P < 0.01; ***P < 0.001).

Table 1 .
Summary (F values) of two-way ANOVA for leaf-to-air vapor pressure deficit (VPD L ) and photosynthetic responses of Larix kaempferi and Pinus densiflora seedlings to temperature and precipitation manipulation.Asterisks show the significant differences to the control (*P < 0.05; **P < 0.01; ***P < 0.05).VPD L , Leaf-to-air vapor pressure deficit; g s , Stomatal conductance; E, Transpiration rate; C i /C a , Ratio of intercellular to ambient CO 2 concentration; P n , Net photosynthetic rate; WUE, Water use efficiency; iWUE, Intrinsic water use efficiency; Chl a , Chlorophyll a content; Chl b , Chlorophyll b content; Chl t , Total chlorophyll content.

Figure 6 .
Figure 6.Transpiration rate (E), ratio of intercellular to ambient CO 2 concentration (C i /C a ), and net photosynthetic rate (P n ) as a function of stomatal conductance (g s ) of Larix kaempferi (a-c) and Pinus densiflora (d-f) seedlings under temperature and precipitation manipulation.Colors of points are represented by canopy temperature (CT).Solid and dashed lines represent the significant and non-significant regressions, respectively.

Table 2 .
Mean (SE) chlorophyll contents of Larix kaempferi and Pinus densiflora seedlings under temperature and precipitation manipulation.Chl a , Chlorophyll a content; Chl b , Chlorophyll b content; Chl t , Total chlorophyll content; TC, Temperature control; T3, + 3 °C Treatment; T6, + 6 °C Treatment; DR, Drought treatment; PC, Precipitation control; HR, Heavy rainfall treatment.Different letters mean the statistical significance among treatments.